Involvement of LEU13 in interferon-induced refractoriness of human RSa cells to cell killing by X rays

Culture of human cells with human interferon alpha and beta (IFNA and IFNB) results in increased resistance of the cells to cell killing by X rays. To identify candidate genes responsible for the IFN-induced X-ray resistance, we searched for genes whose expression levels are increased in human RSa cells treated with IFNA, using an mRNA differential display method and Northern blotting analysis. RSa cells, which showed increased survival (assayed by colony formation) after X irradiation when they were treated with IFNA prior to irradiation, showed increased expression levels of LEU13 (IFITM1) mRNA after IFNA treatment alone. In contrast, IF(r) and F-IF(r) cells, both of which are derived from RSa cells, showed increased X-ray resistance and high constitutive LEU13 mRNA expression levels compared to the parental RSa cells. Furthermore, the IFNA-induced resistance of RSa cells to killing by X rays was suppressed by antisense oligonucleotides for LEU13 mRNA. LEU13, a leukocyte surface protein, was previously reported to mediate the actions of IFN such as inhibition of cell proliferation. The present results suggest a novel role of LEU13 different from that in the inhibition of cell proliferation, involved in IFNA-induced refractoriness of RSa cells to X rays.     

Mitochondrial membrane potential and intracellular ATP content after transient experimental ischemia in the cultured hippocampal neuron

Ischemia limits the delivery of oxygen and glucose to cells and disturbs the maintenance of mitochondrial membrane potential (MMP). MMP regulates the production of high-energy phosphate and apoptotic cascading. Thus, MMP is an important parameter determining the fate of neurons. Differences in the time course of MMP according to the grading of the ischemic impact have not been clarified. MMP and intracellular ATP contents were monitored before and after short-term oxygen-glucose deprivation. A primary hippocampal culture seeded in a 35 mm fenestrated dish for fluorescence microscopy was mounted in a sealed chamber for an anaerobic incubation. A continuous flow of 100% nitrogen into the chamber and a replacement of glucose-free medium allowed the condition of oxygen-glucose deprivation (OGD), thereby extrapolating ischemia. MMP was evaluated by the fluorescence of a voltage-dependent dye, JC-1, under fluorescence microscopy. The intracellular ATP content was evaluated in a hippocampal culture seeded in a 96-well plate by the luciferin-luciferase reaction after a designated period of OGD. During OGD, MMP decreased to 0.72+/-0.03 (normalized JC-1 fluorescence), then increased to the hyperpolarized level 1.99+/-0.12 during 60 min reoxygenation after 30 min OGD. MMP after 60 min OGD decreased and recovered occasionally during reoxygenation. After 90 min OGD and reoxygenation, MMP was reduced and never recovered. The intracellular ATP content was 8.1+/-6.6 and 3.2+/-1.9% after 30 min OGD and 30 min reoxygenation following 30 min OGD, respectively; 60 min OGD did not significantly change these levels (7.1+/-5.8, 2.6+/-0.5%). Hyperpolarization after OGD did not accompany ATP production. This observation suggests the inhibition of electron reentry into an inner membrane during reoxygenation and the disturbance of FoF1-ATP synthase. This pathological finding of an energy-producing system after OGD may provide a clue to explain post-ischemic energy failure.     

Increased response of liver microsomal delta 6-desaturase activity to dietary methionine in rats

The effects of dietary casein level (5-40%) on the liver microsomal phospholipid profile, delta 6-desaturase activity and related variables were investigated in rats to examine whether the dietary protein level affected the delta 6-desaturase activity through an alteration of the liver microsomal phospholipid profile. The effects of supplementing a 10% casein diet with certain amino acids were also investigated. The concentration of hepatic S-adenosylmethionine (SAM), the ratio of phosphatidylcholine (PC) to phosphatidylethanolamine (PE) and the delta 6-desaturase activity in liver microsomes, and the ratio of arachidonate to linoleate of microsomal PC increased with increasing dietary casein level. There were significant correlations between the dietary methionine content and hepatic SAM concentration, hepatic SAM concentration and microsomal PE concentration, and microsomal PE concentration and delta 6-desaturase activity. Supplementation of the 10% casein diet with methionine significantly increased the hepatic SAM concentration, PC/PE ratio, delta 6-desaturase activity, and arachidonate/linoleate ratio, whereas cystine supplementation had no or little effect on these variables. These increases induced by methionine were significantly suppressed by additional glycine. The results obtained here, together with those in our previous report, suggest that quantity and type of dietary protein might affect the delta 6-desaturase activity through an alteration of the liver microsomal profile of phospholipids, especially PE, and that the alteration of phospholipid profile might be mediated by a hepatic SAM concentration that reflects the dietary methionine level.     

Search for UV-responsive genes in human cells by differential mRNA display: involvement of human ras-related GTP-binding protein, Rheb, in UV susceptibility

We have previously demonstrated that glucose deprivation alters the glycosylation of the GLUT1 glucose transporter in 3T3-L1 adipocytes. Many aberrantly glycosylated proteins are retained in the endoplasmic reticulum by interaction with chaperones. Herein, we use three independent procedures to show that GLUT1 is targeted to the plasma membrane, despite alterations in glycosylation. While earlier experiments revealed that plasma membrane targeting of aglyco GLUT 1 transporter was significantly reduced, our data show for the first time that altered glycosylation provides sufficient information to drive appropriate trafficking.     

Influence of temperature on activity of the isolated whole bladder preparation of neonatal and adult rats

The temperature sensitivity of in vitro whole bladder preparations from neonatal and adult rats with or without chronic partial urethral obstruction was investigated. After the bladder was filled to a volume eliciting isovolumetric contractions, temperature was changed between 19 and 38 degrees C. In all preparations, higher temperatures were associated with higher frequencies of spontaneous intravesical pressure waves (IVPW). In 1- to 2-wk-old neonates, IVPW amplitude increased as the temperature increased; however, in older neonates and normal adults, the opposite occurred. The transition period was at 3 wk of age when bladder volume also markedly increased. At this age as well as in adult rats with outlet obstruction, changing temperature had little influence on the amplitude of IVPW. Thus obstructed outlet bladders and 3-wk-old bladders had similar properties. It is concluded that the properties of bladder muscle are changed during postnatal maturation and that in 3-wk-old rats, when brain control of voiding is emerging, micturition is abnormal, leading to obstructive changes in bladder muscle.     

Search for gravity-responsive genes by PCR-based mRNA differential display in human cells

Mechanisms of molecular responses of human cells to gravity change and/or space radiation are one of the most important physiological problems in space science. We have previously reported that expression levels of several genes are changed in cultured human cells after UVC irradiation, and a few of those genes are responsible for UVC sensitivity. In this study, to find candidates for genes that play roles in susceptibility of human cells to gravity stressors, including those responsible for genetic stability in humans, we analyzed genes expressed differentially after gravity stress in human cells, using a PCR-based mRNA differential display (D.D.) method. Cells used were RSa and its variant cell lines, with discrepant sensitivity to radiation cell-killing and mutagenicity [correction of mutagenecity].     

Organic matter processing by chironomid larvae (Diptera: Chironomidae)

In laboratory experiments, we used fluorescent dye markers to investigate processing of organic matter by larvae of Psectrocladius limbatellus (Holm.) (Diptera: Chironomidae). 59% of the organic matter used was incorporated into tubes, 39% was present in faecal pellets (both after 24 h), and 2% was found in the larval gut at the end of experiments. Ingested matter passed through the gut rapidly, resulting in the gut being emptied more than 20 times each day. Further 24-h experiments using dye-marked faecal pellets showed that 6% of pellets produced were re-ingested and 12% were incorporated into tubes. There was no preference for conditioned faecal pellets as food over those that had recently been egested and tubes also provided a food reserve on which larvae feed. Chironomid larvae recycle organic matter resulting in its mineralization and their ‘engineering’ has a dramatic effect on the substratum. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effectiveness of Trivalent Inactivated Influenza Vaccine in Children Estimated by a Test-Negative Case-Control Design Study Based on Influenza Rapid Diagnostic Test Results

We assessed vaccine effectiveness (VE) against medically attended, laboratory-confirmed influenza in children 6 months to 15 years of age in 22 hospitals in Japan during the 2013–14 season. Our study was conducted according to a test-negative case-control design based on influenza rapid diagnostic test (IRDT) results. Outpatients who came to our clinics with a fever of 38°C or over and had undergone an IRDT were enrolled in this study. Patients with positive IRDT results were recorded as cases, and patients with negative results were recorded as controls. Between November 2013 and March 2014, a total of 4727 pediatric patients (6 months to 15 years of age) were enrolled: 876 were positive for influenza A, 66 for A(H1N1)pdm09 and in the other 810 the subtype was unknown; 1405 were positive for influenza B; and 2445 were negative for influenza. Overall VE was 46% (95% confidence interval [CI], 39–52). Adjusted VE against influenza A, influenza A(H1N1)pdm09, and influenza B was 63% (95% CI, 56–69), 77% (95% CI, 59–87), and 26% (95% CI, 14–36), respectively. Influenza vaccine was not effective against either influenza A or influenza B in infants 6 to 11 months of age. Two doses of influenza vaccine provided better protection against influenza A infection than a single dose did. VE against hospitalization influenza A infection was 76%. Influenza vaccine was effective against influenza A, especially against influenza A(H1N1)pdm09, but was much less effective against influenza B.     

Protein disulfide isomerase-P5, down-regulated in the final stage of boar epididymal sperm maturation,catalyzes disulfide formation to inhibit protein function in oxidative refolding of reduced denatured lysozyme

In mammalian spermiogenesis, sperm mature during epididymal transit to get fertility. The pig sharing many physiological similarities with humans is considered a promising animal model in medicine. We examined the expression profiles of proteins from boar epididymal caput, corpus, and cauda sperm by two-dimensional gel electrophoresis and peptide mass fingerprinting. Our results indicated that protein disulfide isomerase-P5 (PDI-P5) human homolog was down-regulated from the epididymal corpus to cauda sperm, in contrast to the constant expression of protein disulfide isomerase A3 (PDIA3) human homolog. To examine the functions of PDIA3 and PDI-P5, we cloned and sequenced cDNAs of pig PDIA3 and PDI-P5 protein precursors. Each recombinant pig mature PDIA3 and PDI-P5 expressed in Escherichia coli showed thiol-dependent disulfide reductase activities in insulin turbidity assay. Although PDIA3 showed chaperone activity to promote oxidative refolding of reduced denatured lysozyme, PDI-P5 exhibited anti-chaperone activity to inhibit oxidative refolding of lysozyme at an equimolar ratio. SDS-PAGE and Western blotting analysis suggested that disulfide cross-linked and non-productively folded lysozyme was responsible for the anti-chaperone activity of PDI-P5. These results provide a molecular basis and insights into the physiological roles of PDIA3 and PDI-P5 in sperm maturation and fertilization.